For peptide isoelectric focusing (IEF) separation, the resulting peptide mixture (1.2mg in total) was resuspended in a buffer containing 6% glycerol and 1.2% ampholytes in the 3-10 pH linear OFFGEL buffer (7M Urea, 2M thiourea, 1% DTT w/v) (GE Healthcare, Uppsala, Sweden). Sample volumes of 150 μl were loaded onto a commercially available 24-cm IPG strip with a linear 3-10 pH gradient (GE Healthcare) after rehydration of the gel for 20 min in a well of 40 μl rehydration solution. Cover fluid (mineral oil, Agilent Technologies) was applied to both ends of the gel strip. Electrofocusing of the peptides was performed at 20°C and 50 μA until 50 kVh were reached using an Agilent 3100 OFFGEL fractionator (Agilent Technologies) following the manufacturer instructions. Fractions were recovered, peptides extracted from each well with 2% TFA (v/v) and desalted by passing through a home-made column packed with Poros 50 R2 resin (Applied Biosystem, Foster City, CA, USA). Peptides were eluted with 50% ACN (v/v) in 0.1% TFA (v/v) and the fractions were dried and reconstituted in 0.1% formic acid (v/v) just before LC-MS analysis.
24 cm ipg strip
Manufactured by GE Healthcare
Sourced in United States, Sweden
The 24 cm IPG strips are a laboratory instrument used for protein separation and analysis. They provide a consistent and reliable platform for isoelectric focusing, a key technique in two-dimensional gel electrophoresis. The strips are designed to deliver accurate and reproducible results in proteomics research and biomarker discovery applications.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 3100 offgel fractionator, by Agilent Technologies (2 mentions)
2 de manual, by GE Healthcare (2 mentions)
Mineral oil, by Agilent Technologies (1 mentions)
Ettan dalt system, by GE Healthcare (1 mentions)
Image master 2d platinum, by GE Healthcare (1 mentions)
3100 offgel fractionator, by Agilent Technologies (1 mentions)
Ettan dalt six gel system, by GE Healthcare (1 mentions)
Ettan ipgphor 2, by GE Healthcare (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Typhoon trio scanner, by GE Healthcare (1 mentions)
Imagemaster 2d platinum software, by GE Healthcare (1 mentions)
Ettan ipgphor isoelectric focusing system, by GE Healthcare (1 mentions)
Pdquest 2 d analysis software, by Bio-Rad (1 mentions)
5800 maldi tof tof mass spectrometer, by AB Sciex (1 mentions)
Imagescanner 3, by GE Healthcare (1 mentions)
12 protocols using 24 cm ipg strip
1
Peptide Isoelectric Focusing Separation
2
Two-dimensional Gel Electrophoresis Proteomics
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Approximately 1,300 µg of the protein samples were diluted to a volume of 450 µL with lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, 13 mM DTT), followed by loading onto a 24-cm IPG strip (immobilized pH gradient) with a linear pH gradient of 4–7 (GE Healthcare, Uppsala, Sweden). The strips were hydrated for 18 h at room temperature, and then subjected to IEF on an Ettan IPGphor isoelectric focusing system, following the instructions of the manufacturer (2-DE Manual, GE Healthcare), with some modifications as described previously22 (link). After IEF, the IPG strips were equilibrated first in an equilibration solution containing 1% DTT, followed by an equilibration solution with 4% iodoacetamide. The strips were then transferred to an Ettan Dalt system (GE Healthcare) to perform SDS-PAGE using 12.5% SDS polyacrylamide gels47 (link).
The gels were visualized via the GAP staining method48 (link) and scanned with ImageMaster Labscan V3.0 (GE Healthcare, Uppsala, Sweden), and image analysis was performed with the ImageMaster 2D Platinum software package (GE Healthcare, Uppsala, Sweden). Spots that were present in all replicate gels, showing Student’s t test p-values <0.05 and a relative fold change of at least a 1.5 in their quantity, were further analyzed.
The gels were visualized via the GAP staining method48 (link) and scanned with ImageMaster Labscan V3.0 (GE Healthcare, Uppsala, Sweden), and image analysis was performed with the ImageMaster 2D Platinum software package (GE Healthcare, Uppsala, Sweden). Spots that were present in all replicate gels, showing Student’s t test p-values <0.05 and a relative fold change of at least a 1.5 in their quantity, were further analyzed.
3
Protein Fractionation by OFFGEL Isoelectric Focusing
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The purified protein sample (100 μg) from each patient was prepared in a 3.6-mL sample volume and fractionated by a 3100 OFFGEL fractionator (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. The 24-cm IPG strip (pH 3-10, GE Healthcare, Bio-Sciences) was fixed on a 24-well loading tray. The IPG strip was rehydrated with 40 μL of OFFGEL stock solution for 15 minutes. Then, 150 μL of sample volume was loaded into each well and run for a total power of 64 KVhr using a constant electric current of 50 μA. After focusing, the separated proteins in 24 fractions were purified with cold (−20°C) acetone at −20°C overnight and centrifuged at 12 000× g at 4°C for 10 minutes. The protein pellets were dissolved in a solution containing 8 M urea and 2 M thiourea and quantitated by Bradford assay.
4
Protein Separation by 2D-PAGE
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A 1 mg protein sample was loaded on a 24 cm IPG strip (immobilized pH gradient, pH 4–7, linear, GE Healthcare) (Amersham Bioscience, Uppsala, Sweden). Each protein sample was assessed in triplicate. Isoelectric focusing (IEF) was carried out at 20 °C for 14 h at 30 V, 2 h at 200 V, 0.5 h at 500 V, 1 h at 1000 V, 3 h at 8000 V, and then held at 8000 V until a total of at least 60 000 Vh was reached (Ettan IPGphorII, GE Healthcare, Uppsala, Sweden). Focused IPG strips were equilibrated for 15 min in equilibration buffer (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris pH 8.8, 1% DTT) under gentle agitation, and then for an additional 15 min in the same buffer, except that DTT was substituted with 2.5% iodoacetamide. After equilibration, the strips were transferred to vertical slab gels (12% SDS–PAGE) for second-dimensional electrophoresis with the Ettan DALT six gel system (GE Healthcare).
5
iTRAQ Peptide Desalting and Fractionation
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Peptides from iTRAQ pools were desalted using Sep-Pak C18 columns, eluted with 60% ACN and dried in a SpeedVac system. Focusing was performed on Agilent 3100 Offgel Fractionator (Agilent Technologies, Palo Alto, USA) according to the manufacturer’s protocol. After 1 h of rehydration with a voltage of 500 V, peptides were focused until 80 kVh was reached on 24 cm IPG strip (GE Healthcare, Milwaukee, USA) with a 4–7 pH range. The 24-well tray was used and the collected fractions were cleaned with Sep-Pak C18 columns, dried with a SpeedVac system and stored at -20°C until the MS analyses were performed.
6
Two-Dimensional Gel Electrophoresis of CML-T1 Cells
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Isoelectric focusing was performed with a Bio-Rad Protean IEF cell using 24 cm IPG strips (pH 4.0–7.0; GE Healthcare Life Sciences). Six technical replicates were run for each biological sample (6x CML-T1 and 6x CML-T1/IR). The strips were rehydrated overnight, each in 450 μl of sample, representing 3.3 mg of protein. Isoelectric focusing was performed for 60 kVh, with the maximum voltage not exceeding 5 kV, the current limited to 50 mA/strip and the temperature set to 18°C. The focused strips were equilibrated and reduced in equilibration buffer A (6 M urea, 50 mM Tris pH 8.8, 30% glycerol, 2% SDS and 450 mg DTT/50 ml of the buffer) for 15 min and then alkylated in equilibration buffer B (6 M urea, 50 mM Tris pH 8.8, 30% glycerol, 2% SDS and 1.125 mg iodacetamide/50 ml). The equilibrated strips were then placed on the top of 10% PAGE gel and secured in place by molten agarose. Electrophoresis was performed in a Tris-glycine-SDS system using a Protean Plus Dodeca Cell apparatus (Bio-Rad, Inc.) with buffer circulation and external cooling (20°C). The twelve gels were run at a constant voltage of 200 V for 6 h. Following electrophoresis, the gels were washed three times for 15 min in deionized water to remove the SDS. The washed gels were stained in CCB (SimplyBlue SafeStain, Invitrogen Life Technologies, Carlsbad, CA, USA) overnight, and then destained in deionized water.
7
2D DIGE Analysis of Microsomal Proteins
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2D DIGE was performed as described elsewhere.22 (link) Twenty micrograms of microsomal proteins from
dark-control or blue light-treated plants (pH 8.5) were mixed with
80 pmol of Cy3 or Cy5 dyes and incubated on ice for at least 2 h in
the dark. The labeling reaction was terminated by adding 0.5 μL
of 10 mM lysine. Cy3- and Cy5-labeled proteins were then combined
and used for isoelectric focusing (IEF). IEF was performed on 24 cm
IPG strips, pH 3–10 NL (GE Healthcare, Piscataway, NJ, USA).
The running conditions were as follows: rehydration for 2 h, 50 V
for 10 h, step and hold at 500 V and then 1000 V for 1 h each, gradient
to 8000 V over 3 h, and then at 8000 V until reaching a total of 56 000
V h. Second-dimension electrophoresis was performed using 10% SDS-polyacrylamide
gels. The electrophoresis was performed at 40 V for 2 h and then at
120 V until the bromophenol blue front reached the bottom of the gel.
Images of Cy3- and Cy5-labeled proteins were acquired using a Typhoon
Trio scanner (GE Healthcare). The estimated pH ranges following IEF
are indicated in Figures2 –4 .
dark-control or blue light-treated plants (pH 8.5) were mixed with
80 pmol of Cy3 or Cy5 dyes and incubated on ice for at least 2 h in
the dark. The labeling reaction was terminated by adding 0.5 μL
of 10 mM lysine. Cy3- and Cy5-labeled proteins were then combined
and used for isoelectric focusing (IEF). IEF was performed on 24 cm
IPG strips, pH 3–10 NL (GE Healthcare, Piscataway, NJ, USA).
The running conditions were as follows: rehydration for 2 h, 50 V
for 10 h, step and hold at 500 V and then 1000 V for 1 h each, gradient
to 8000 V over 3 h, and then at 8000 V until reaching a total of 56 000
V h. Second-dimension electrophoresis was performed using 10% SDS-polyacrylamide
gels. The electrophoresis was performed at 40 V for 2 h and then at
120 V until the bromophenol blue front reached the bottom of the gel.
Images of Cy3- and Cy5-labeled proteins were acquired using a Typhoon
Trio scanner (GE Healthcare). The estimated pH ranges following IEF
are indicated in Figures
8
Proteomic Analysis of SH-SY5Y Cells
9
2-DE Protein Profiling Workflow
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2-DE was performed on an Ettan IPGphor isoelectric focusing system according to the manufacturer’s instructions (2-DE Manual, GE Healthcare, Uppsala, Sweden). The 24 cm IPG strips (immobilized pH gradient) with a linear pH gradient of 4–7 (GE Healthcare) were used, approximately 1,300 µg protein samples were loaded on, and 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gels were used for SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Each protein extracts were performed on 2-DE gels in triplicate for technical replicates. The experimental procedures were as previously described30 (link).
Gels were stained using a GAP staining method46 (link) and scanned with the ImageMaster Labscan V3.0 (GE Healthcare). Image analysis was conducted using a ImageMaster 2D Platinum software package (GE Healthcare). Only the spots that were present in all replicate gels and shown a Student’s t test p-value < 0.05 and a relative change in quantity of at least 1.5-fold in their quantity, were considered as DAPs for further analysis30 (link).
Gels were stained using a GAP staining method46 (link) and scanned with the ImageMaster Labscan V3.0 (GE Healthcare). Image analysis was conducted using a ImageMaster 2D Platinum software package (GE Healthcare). Only the spots that were present in all replicate gels and shown a Student’s t test p-value < 0.05 and a relative change in quantity of at least 1.5-fold in their quantity, were considered as DAPs for further analysis30 (link).
10
Proteomic Profiling of 2KGA Production
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Sample preparation was performed at the middle stages of 2KGA production phase and the consumption phase. Cells were harvested by centrifugation (10,000 × g, 1 min, and 4 °C) and immediately frozen in liquid nitrogen. The whole-cell protein extraction was performed according to reference [30 (link)]. Proteins were separated by two-dimensional gel electrophoresis (2-DE). For isoelectric focusing, 1 mg of proteins were diluted in the immobilized pH gradient (IPG) strip rehydration buffer (450 μL). The protein solution was loaded on 24 cm IPG strips (GE Healthcare) that provided a linear gradient from pH 3 to 10, according to the manufacturer’s instructions. Isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis were carried out according to references [30 (link),31 (link)]. Stained 2-DE gels were scanned using an ImageScanner III (GE Healthcare) and analyzed by PDQuest 2-D Analysis Software (Bio-Rad). Protein spots of interest were cut from the gels and trypsin digested in-gel, as described, and identified by MALDI-TOF/TOF tandem MS using an AB SCIEX 5800 MALDI TOF/TOF mass spectrometer (AB SCIEX, USA). MS data were analyzed using the MASCOT 2.1.0 program (Matrix Science, MA), as described [30 (link),31 (link),32 (link)].
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